Control of gene expression in plants by receptor mediated transactivation in the presence of a chemical ligand

ABSTRACT

The present invention is drawn to a method of controlling gene expression in plants. Specifically, the method comprises obtaining a transgenic plant comprising at least two receptor expression cassettes and at least one target expression cassette. The first receptor expression cassette comprises a nucleotide sequence for a 5′ regulatory region operably linked to a nucleotide sequence which encodes a first receptor polypeptide, and a 3′ termination region. The second receptor expression cassette comprises a nucleotide sequence for a 5′ regulatory region operably linked to a nucleotide sequence which encodes a second receptor polypeptide, and a 3′ termination region. The target expression cassette comprises a nucleotide sequence for a 5′ regulatory region operably linked to a nucleotide sequence which encodes a target polypeptide, and a 3′ termination region, wherein the 5′ regulatory region of the target expression cassette is activated by the first and second receptor polypeptides in the presence of one or more chemical ligands which are complementary to the ligand binding domain of the receptor polypeptides, whereby expression of the target polypeptide is accomplished. The method is useful for controlling various traits of agronomic importance, such as plant fertility.

This is a continuation of Application No. 09/625,904, filed Jul. 26,2000, which is a continuation of Application No. 09/234,190, filed Jan.20, 1999, now U.S. Pat. No. 6,147,282, which is a division ofApplication No. 09/010,050, filed Jan. 21, 1998, now U.S. Pat. No.5,880,333, which is a continuation of Application No. 08/398,037, filedMar. 3, 1995, abandoned.

FIELD OF THE INVENTION

The present invention relates to the chemical control of gene expressionin plants. In particular, it relates to a method whereby receptorpolypeptides in the presence of an appropriate chemical ligand regulatethe expression of a target polypeptide in a plant cell, as well as tothe expression cassettes encoding the receptor and target polypeptidesand transgenic plants containing the expression cassettes.

BACKGROUND OF THE INVENTION

In some cases it is desirable to control the time or extent ofexpression of a phenotypic trait in plants, plant cells or plant tissue.An ideal situation would be the regulation of expression of such a traitat will, triggered by a chemical that could be easily applied to fieldcrops, ornamental shrubs, etc. One such system of regulating geneexpression which could be used to achieve this ideal situation, as yetunknown to be present naturally in plants, is the steroid and thyroidhormone superfamily of nuclear receptors.

The steroid and thyroid hormone superfamily of nuclear receptors isfound in mammals and insects and is composed of over 100 known proteins.These receptors fall into at least two functionally distinct categoriesknown as Class I and Class II. Beato, Cell 56: 335-344(1989); Parker,Sem. Cancer Biol. Ser. 1: 81-87(1990). Of the two classes, only theClass II receptors function in the nucleus as heterodimers to affectexpression of target genes in the presence of hormone. The best studiedexamples of Class II receptor proteins are Retinoic Acid Receptor (RAR),Vitamin D Receptor (VDR), and Thyroid Hormone Receptor (T₃R) andRetinoic X Receptor (RXR). The receptors bind to the 5′ regulatoryregion of the target gene and, upon binding of a chemical ligand to thereceptor, the transcriptional activation (transactivation) domain of thereceptor affects gene expression by interacting with other transcriptioninitiating factors.

In addition to the Class II receptor proteins found in mammals asdescribed above, receptors of similar structure and activity have beenindentified in the insect Drosophila. Koelle et al., Cell 67: 59 (1991);Christianson and Kafatos, Biochem. Biophys. Res. Comm. 193: 1318 (1993);Henrich et al., Nucleic Acids Res. 18: 4143 (1990). The EcdysoneReceptor (EcR) binds the steroid hormone 20-hydroxyecdysone and, whenheterodimerized with the product of the Ultraspiracle gene (USP), willtransactivate gene expression. USP is most homologous to RXRα, and RXRis capable of forming heterodimers with EcR. Thomas et al., Nature 362:471-475 (1993). Additional chemical ligands besides 20-hydroxyecdysone,such as other hormone agonists or antagonists, will also bind to thesereceptors and cause transactivation of a target gene.

One member of the steroid and thyroid superfamily of nuclear receptors,the Class I Glucocorticoid Receptor (GR) which utilizes chaperonins anddoes not function by heterodimerization with other receptors, has beenshown to transactivate a target gene in plant cells. Schena et al.,Proc. Natl. Acad. Sci. USA 88: 10421-10425 (1991). A fragment containingthe ligand binding domain from GR was fused to the anthocyaninregulatory protein known as ‘R’ and shown to stimulate production ofanthocyanin in transgenic Arabidopsis thaliana in response to theapplication of the appropriate chemical ligand. Lloyd et al., Science226: 436 (1994). It was also reported by Lloyd et al. that full-lengthGR did not activate gene expression in stably transformed Arabidopsisthaliana whereas it did in transient assays in tobacco protoplasts.Furthermore, fusions of R with a fragment from the Estrogen Receptor(ER), another Class I receptor which utilizes chaperonins, alsostimulated production of anthocyanin in the presence of the appropriatechemical ligand but showed ‘substantial’ background expression.

The distinguishing feature of the Class II receptor proteins,transactivation of a target gene by heterodimerized receptors in thepresence of an appropriate chemical ligand, offer previouslyunrecognized opportunities for chemical control of gene expression inplants. The use of heterodimers allows a broader range of gene controlstrategies, and chemicals are already known for agricultural use whichcan trigger receptor-mediated transactivation of target gene expressionof this class. Furthermore, gene control strategies for plants whichutilize nuclear receptors that do not occur naturally in plants have theattractive feature of inducing only the genetically engineered targetgene. The class II receptors in general, however, possess fairly poortranscriptional activation domains, and the ability of the receptors totransactivate target genes may be enhanced by the addition of othertranscriptional activation domains, particularly from plant or viralspecies. Further modification would also be needed in order to provideminimum basal activity which increases rapidly to high levels in thepresence of a triggering chemical. As has been demonstrated by thepresent invention, receptor polypeptides based on the class II model,and the genes that encode them, have been developed which function inplant cells to control expression of a target polypeptide wherein thereceptor polypeptides activate the 5′ regulatory region of a targetexpression cassette in the presence of a chemical ligand. Such a methodof controlling gene expression in plants would be useful for controllingvarious traits of agronomic importance, such as plant fertility.

SUMMARY OF THE INVENTION

The present invention is drawn to a method of controlling geneexpression in plants. Specifically, the method comprises transformning aplant with at least two receptor expression cassettes and at least onetarget expression cassette. The first receptor expression cassettecomprises a nucleotide sequence for a 5′ regulatory region operablylinked to a nucleotide sequence which encodes a first receptorpolypeptide operably linked to a 3′ termination region. The secondreceptor expression cassette comprises a nucleotide sequence for a 5′regulatory region operably linked to a nucleotide sequence which encodesa second receptor polypeptide operably linked to a 3′ terminationregion. The first and second receptor polypeptides comprise a first andsecond ligand binding domain, respectively, which are mutually distinct.The target expression cassette comprises a nucleotide sequence for a 5′regulatory region operably linked to a nucleotide sequence which encodesa target polypeptide operably linked to a 3′ termnination region,wherein the 5′ regulatory region of said target expression cassette isactivated by said first and second receptor polypeptides in the presenceof one or more chemical ligands, whereby expression of said targetpolypeptide is accomplished. The method is useful for controllingvarious traits of agronomic importance, such as plant fertility.

The invention is further drawn to transgenic plants comprising a firstand second receptor expression cassette and a target expressioncassette. Also encompassed by the invention are receptor expressioncassettes and target receptor cassettes capable of high level expressionin plants.

The receptor polypeptides comprise a ligand binding domain, DNA bindingdomain and a transactivation domain. Further, the receptor polypeptidesmay have a chimeric form, where one or more of the ligand binding, DNAbinding or transactivation domains are obtained from a sourceheterologous with respect to other domains present in the chimericreceptor polypeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Pictoral representation of a plant cell comprising a firstreceptor expression cassette which encodes a first receptor polypeptidecomprising a first ligand binding domain, and a second receptorexpression cassette encoding a second receptor polypeptide comprising asecond ligand binding domain. The first and second receptor polypeptidesare mutually distinct. The plant cell further comprises a targetexpression cassette which encodes a target polypeptide, wherein thetarget polypeptide is expressed upon activation of the 5′ regulatoryregion of the target expression cassette by the first and secondreceptor polypeptides in the presence of a chemical ligand which iscomplementary to said first or second ligand binding domain.

FIG. 2. Same as FIG. 1, except the first receptor polypeptide is theEcdysone Receptor (EcR), the second receptor polypeptide isUltraspiracle (USP), and the chemical ligand is MIMIC®.

FIG. 3. Same as FIG. 1, except the first receptor polypeptide is aGAL4-EcR fusion, the second receptor polypeptide is a USP-VP16 fusion,and the chemical ligand is MIMIC®.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and Nomenclature

“Receptor polypeptide” as used herein refers to polypeptides whichactivate the expression of a target polypeptide in response to anapplied chemical ligand. The receptor polypeptide is comprised of aligand binding domain, a DNA binding domain and a transactivationdomain. A “receptor expression cassette” comprises a nucleotide sequencefor a 5′ regulatory region operably linked to a nucleotide sequencewhich encodes a receptor polypeptide and an untranslated 3′ terminationregion (stop codon and polyadenylation sequence).

The ligand binding domain comprises a sequence of amino acids whosestructure binds non-covalently a complementary chemical ligand. Hence, aligand binding domain and its chemical ligand form a complementarybinding pair.

The DNA binding domain comprises a sequence of amino acids which bindsnon-covalently a specific nucleotide sequence known as a responseelement (RE). The response elements are located in the 5′ regulatoryregion of the target expression cassette and comprise a pair ofhalf-sites, each half-site having a 6 base pair core where a single DNAbinding domain recognizes a single half-site. The half-sites may bearranged in relative linear orientation to each other as either directrepeats, palindromic repeats or inverted repeats. A response elementbinds either a homodimer or heterodimer of receptor polypeptides. Thenucleotide sequence and linear orientation of the half-sites determineswhich DNA binding domain or DNA binding domains will form acomplementary binding pair with said response element, as well as theability of receptor polypeptides to interact with each other in a dimer.

The transactivation domain comprises one or more sequences of aminoacids acting as subdomains which affect the operation of transcriptionfactors during preinitiation and assembly at the TATA box. The effect ofthe transactivation domain is to allow repeated transcription initiationevents, leading to greater levels of gene expression.

A “moiety” refers to that share or portion of a receptor polypeptidethat is derived from the indicated source. For example, “EcR-moiety”refers to that portion of the receptor polypeptide that was derived fromthe native ecdysone receptor. Moiety as used here may comprise one ormore domains.

“Homologous” is used to indicate that a receptor polypeptide has thesame natural origin with respect to its current host. For example, theecdysone receptor (EcR) is found in certain insect species and is saidto be homologous with respect to the insect species in which itoriginates. “Homologous” is also used to indicate that one or more ofthe domains present in a receptor polypeptide have the same naturalorigin with respect to each other. For example, the DNA binding domainand the ligand binding domain of EcR are considered to be of ahomologous origin with respect to each other.

“Heterologous” is used to indicate that a receptor polypeptide has adifferent natural origin with respect to its current host. For example,if the ecdysone receptor (EcR) from an insect species is expressed in aplant cell, then the EcR is described as being heterologous with respectto its current host, which is the plant cell. “Heterologous” is alsoused to indicate that one or more of the domains present in a receptorpolypeptide differ in their natural origin with respect to other domainspresent. For example, if the transactivation domain from the herpessimplex VP16 protein is fused to the USP receptor from Drosophila, thenthe VP16 transactivation domain is heterologous with respect to theUSP-moiety. Furthermore, if a domain from USP is fused to a domain fromRXR to make a functional receptor, then the chimeric fusion would havedomains that are heterlogous to each other. In addition, a heterologousreceptor polypeptide comprising the fusion of a VP16 transactivationdomain and a USP-moiety, when expressed in a plant, would also beconsidered heterologous with respect to the plant host.

The term “chimeric” is used to indicate that the receptor polypeptide iscomprised of domains at least one of which has an origin that isheterologous with respect to the other domains present. These chimericreceptor polypeptides are encoded by nucleotide sequences which havebeen fused or ligated together resulting in a coding sequence which doesnot occur naturally.

The chimeric receptor polypeptides of the present invention arereferenced by a linear nomenclature from N-terminal to C-terminalportion of the polypeptide. Using this nomenclature, a chimeric receptorpolypeptide having the transactivation domain from VP16 fused to theN-terminal end of the USP receptor would be designated as VP16-USP.Conversely, if VP16 was fused to the C-terminus of the USP receptor thechimeric receptor polypeptide would be designated USP-VP16.

Gene constructions are denominated in terms of a 5′ regulatory regionand its operably-linked coding sequence, where the 5′ regulatory regionis designated before a slash mark (/) and the coding sequence designatedafter the slash mark. For example, the gene construction 35USP-VP16designates the 35S promoter of Cauliflower Mosaic Virus fused to thechimeric receptor USP-VP16, where the transactivation domain of VP16 isfused to the C-terminal end of USP.

A “target expression cassette” comprises a nucleotide sequence for a 5′regulatory region operably linked to a nucleotide sequence which encodesa target polypeptide whose expression is activated by the receptorpolypeptides in the presence of a chemical ligand. The 5′ regulatoryregion of the target gene comprises a core promoter sequence, aninitiation of transcription sequence and the response element orresponse elements necessary for complementary binding of the receptorpolypeptides. The target expression cassette also possesses a 3′termination region (stop codon and polyadenylation sequence).

Native and Chimeric Receptor Polypeptides for Controlling GeneExpression in Plants

The method of the present invention comprises expressing within a plantat least two receptor polypeptides which, in the presence of one or morechemical ligands, activate the 5′ regulatory region of a targetexpression cassette within a transgenic plant (FIG. 1). At least tworeceptor polypeptides are required to activate the 5′ regulatory region.These two receptor polypeptides form a dimer. When the two receptorpolypeptides are identical they form a “homodimer” and when the tworeceptor polypeptides are different they form a “heterodimer.” One orboth of the two receptor polypeptides present in a homodimer orheterodimer may be in a chimeric form, as described below. Examples ofheterodimers encompassed by the invention include, but are not limitedto, EcR+USP, EcR+RXR or chimeric forms thereof.

The receptor polypeptides are composed of a ligand binding domain, a DNAbinding domain and a transactivation domain. The DNA binding domainbinds the receptor polypeptide to the 5′ regulatory region of the targetexpression cassette at the site of the response element. The ligandbinding domain of the receptor polypeptides binds, when present, thecomplementary chemical ligand. Binding of the chemical ligand causes aconformational change in the receptor polypeptide and allows thetransactivation domain to affect transcription of the coding sequence ofthe target expression cassette, resulting in production of the targetpolypeptide.

The chimeric receptor polypeptides used in the present invention haveone or more domains obtained from a heterologous source. The use ofchimeric receptor polypeptides has the benefit of combining domains fromdifferent sources, thus providing a receptor polypeptide activated by achoice of chemical ligands and possessing superior ligand binding, DNAbinding and transactivation characteristics. One preferred embodiment ofthe present invention are chimeric receptor polypeptides where thecomplementary chemical ligand is selected from an insecticide, an insecthormone, or antagonists or agonists of insect hormones.

The 5′ regulatory region of the receptor expression cassettes furthercomprises a promoter which permits expression in plant tissues andcells. Appropriate promoters are chosen for the receptor expressioncassettes so that expression of the receptor polypeptides may beconstitutive, developmentally regulated, tissue specific, cell specificor cell compartment specific. Promoters may also be chosen so thatexpression of the receptor polypeptides themselves can bechemically-induced in the plant, thereby increasing the level ofpromoter induction by ligand. By combining promoter elements whichconfer specific expression with those conferring chemically-inducedexpression, the receptor polypeptides may be expressed or activatedwithin specific cells or tissues of the plant in response to chemicalapplication. The nucleotide sequence which encodes the receptorpolypeptide may be modified for improved expression in plants, improvedfunctionality, or both. Such modifications include, but are not limitedto, altering codon usage, insertion of introns or creation of mutations.In one embodiment of the invention, expression cassettes comprising ananther-specific or pistil-specific promoter operably linked to anucleotide sequence which encodes a chimeric receptor polypeptide areused to activate the expression of a target polypeptide. Targetpolypeptides whose expression is activated by the receptor polypeptidesin the presence of a chemical ligand are also disclosed. The expressionof any coding sequence may be controlled by the present invention,provided that the promoter operably linked to said coding sequence hasbeen engineered to contain the response element or response elementswhich are complementary to the DNA binding domain of the receptorpolypeptides used. For example, target polypeptides which are useful forcontrolling plant fertility are activated by the receptor polypeptidesin the presence of a chemical ligand.

Selecting Domains for Use in a Chimeric Receptor Polypeptide

Chimeric receptor polypeptides may be used in the present invention toactivate expression of a target polypeptide. One or more of the threedomains of a receptor polypeptide may be chosen from a heterologoussource based upon their effectiveness for transactivation, DNA bindingor chemical ligand binding. The domains of the chimeric receptorpolypeptide may also be obtained from any organism, such as plants,insects and mammals which have similar transcriptional regulatingfunctions. In one embodiment of the invention, these domains areselected from other members the steroid and thyroid hormone superfamilyof nuclear receptors. Chimeric receptor polypeptides as provided hereinoffer the advantage of combining optimum transactivating activity,complementary binding of a selected chemical ligand and recognition of aspecific response element. Thus, a chimeric polypeptide may beconstructed that is tailored for a specific purpose. These chimericreceptor polypeptides also provide improved functionality in theheterologous environment of a plant cell.

It is also considered a part of the present invention that thetransactivation, ligand-binding and DNA-binding domains may be assembledin the chimeric receptor polypeptide in any functional arrangement. Forexample, where one subdomain of a transactivation domain is found at theN-termninal portion of a naturally-occuring receptor, the chimericreceptor polypeptide of the present invention may include atransactivation subdomain at the C-terminus in place of, or in additionto, a subdomain at the N-terminus. Chimeric receptor polypeptides asdisclosed herein may also have multiple domains of the same type, forexample, more than one transactivation domain (or two subdomains) perreceptor polypeptide.

The Ligan Binding Domain

The ligand binding domain of the receptor polypeptide provides the meansby which the 5′ regulatory region of the target expression cassette isactivated in response to the presence of a chemical ligand. The ecdysonereceptor (EcR) from Drosophila is one example of a receptor polypeptidewhere complementary chemical ligands have been identified which bind tothe ligand binding domain. The steroid hormone ecdysone triggerscoordinate changes in tissue development that results in metamorphosis,and ecdysone has been shown to bind to EcR. Koelle et al. Cell 67:59-77, 1991. The plant-produced analog of ecdysone, muristerone, alsobinds to the ligand binding domain of EcR. Other chemicals, such as thenon-steroidal ecdysone agonists RH 5849 (Wing, Science 241: 467-469(1988)) and RH 5992 (tebufenozide), the latter known as the insecticideMIMIC®, also will act as a chemical ligand for the ligand binding domainof EcR. The EcR and its ligand binding domain have been found in thepresent invention to be particularly useful for controlling targetpolypeptide expression in plant cells, as described in the examplesbelow.

Another receptor from Drosophila, Ultraspiracle (USP), also known as“2C”, has been isolated and cloned, and its ligand binding domain domainhas been identified. (Henrich et al., Nucleic Acids Research 18:4143-4148 (1990)). USP is most similar to the steroid receptor RXRα,which has as a chemical ligand 9-cis-retinoic acid. USP has also beenshown to form a heterodimer with EcR and regulate the expression of atarget polypeptide in transformed mice kidney cells in response to theapplication of ecdysone. (Evans et al. WO 94/01558). The receptor USPand its ligand binding domain have been found in the present inventionto be particularly useful for controlling target polypeptide expressionin plants, as described in the examples below.

Ligand binding domains for the construction of chimeric receptorpolypeptides may also be obtained from a variety of other sources.Particularly useful sources of ligand binding domains include but arenot limited to Class II receptor proteins of the steroid and thyroidhormone superfamily of nuclear receptors.

The choice of chemical ligand will depend on which ligand bindingdomains are present in the receptor polypeptide. Any chemical compoundwill suffice as long as it is shown to form a complementary binding pairwith the chosen ligand binding domain. When a naturally-occurringcompound is known to form a complementary binding pair with a particularligand binding domain, these known compounds also find use in thepresent invention. Particularly useful sources of ligand binding domainsinclude but are not limited to Class II receptor proteins of the steroidand thyroid hormone superfamily of nuclear receptors. Particularlyuseful chemicals include but are not limited to insecticides which forma complementary binding pair with the ligand binding domain. Suchchemicals include but are not limited to hormones, hormone agonists orhormone antagonists whose function as insecticides can be acscribed totheir binding to native receptor proteins in insects. In addition,chemicals with these hormone or hormone-related properties which areknown as insecticides have the additional benefit of already beingexamined for agricultural production, making such chemicals“ready-to-use” for field application to crops. Useful chemicals withthese properties include but are not limited to fenoxycarb, CGA 59,205,MIMIC®, and RH 5849.

The invention also encompasses ways of reducing background so thatinduction is large relative to the suppressed background expression.Many ligand binding domains will form a complementary binding pair withmore than one chemical ligand. In some cases, these chemical ligandswill bind but have no known function. In other cases, there may bechemical ligands endogenous to the current host in which the receptorpolypeptides are expressed which will bind to the ligand binding domain.In order to avoid endogenous chemical ligands in a heterologous hostfrom binding with the expressed receptor polypeptides andunintentionally affecting expression of the target gene, it may bedesirable to mutate the coding sequence for the receptor polypeptide sothat it recognizes only the chemical ligand applied exogenously. Usefulmethods of mutagenesis are known in the art, such as chemicalmutagenesis or site-directed mutagenesis.

In one method, mutant receptor polypeptides are prepared by PCRmutagenesis of the nucelotide sequence encoding the ligand bindingdomain of EcR or USP. These mutant receptor polypeptides are expressedin a host organism that lends itself to convenient screening andisolation techniques, such as yeast. Screening for mutant receptorpolypeptides that exhibit decreased basal activity and a greater foldinduction in such a host organism will, however, only provide candidatesfor further testing in plant cells, since it is clear from work with theglucocorticoid receptor (GR) that although receptors from the steroidand thyroid hormone superfamily can function in yeast, it is notpredictive of functionality in transgenic plants (Lloyd et al., Science226: 436 (1994)). Further limiting the application of results from yeastis the observation that yeast cells which express GR do not respond tothe commonly used chemical ligand dexamethasone, while this ligand isfunctional in other heterologous systems (Schena et al., Proc. Natl.Acad. Sci. USA 88: 10421-10425 (1991)).

Further testing in plant cells is accomplished by preparing receptorexpression cassettes which encode the mutated receptor polypeptides andtransforming them into plant cells in combination with a targetexpression cassette. The transformed plant cells are tested foractivation of the 5′-regulatory region of the target expression cassetteby the mutant receptor polypeptides in the presence of an appropriatechemical ligand. Mutant receptor polypeptides which produce low basalexpression of a target polypeptide in the absence of chemical ligand andhigh expression of target polypeptide in the presence of an appropriatechemical ligand are useful for controlling gene expression in plants.

Furthermore, heterodimerization in the absence of ligand can also resultin unintentional activation of the 5′ regulatory region of the targetexpression cassette, thereby producing high levels of basal expressionof the target polypeptide. Another region of the ligand binding domainknown as the heptad repeat region is thought to influence the degree ofheterodimerization in the absence of ligand. (Au-Fliegner et al., Mol.Cell. Biol. 13: 5725 (1993)). In one embodiment of the presentinvention, the ninth heptad repeat of the heptad repeat region ismutated using site directed PCR mutagenesis in such a way as to alterthe interaction between subunits of the receptor polypeptideheterodimers in the absence of chemical ligand.

The DNA Binding Domain and its Response Elements

The DNA binding domain is a sequence of amino acids which has certainfunctional features which are responsible for binding of the receptorpolypeptide to a specific sequence of nucleotides, the responseelements, present in the 5′ regulatory region of the target expressioncassette. In one embodiment of the invention, the DNA binding domain isobtained from a Class II nuclear receptor and contains cysteine residuesarranged in such a way that, when coordinated by zinc ions, forms the socalled “zinc-finger” motif. The structure of DNA binding domains for theClass II nuclear receptors is highly conserved from one species toanother, and consequently there is limited variation in the responseelements used to form a complementary binding pair. (Evans, Science 240:889-895 (1988)). Nevertheless, considerable flexibility can beintroduced into the method of controlling gene expression by using theseconserved response elements in other ways. In a preferred embodiment ofthe invention, multiple copies of the appropriate response element areplaced in the 5′ regulatory region, which allows multiple sites forbinding of receptor polypeptide heterodimers resulting in a greaterdegree of activation.

Further flexibility in the gene control method can be achieved bychanging the linear orientation or position of the response elements inthe 5′ regulatory region. The response elements which are recognized byClass II receptor proteins have a “dyad” symmetry, consistent with theirfunctioning with dimerized receptor polypeptides to control geneexpression. (Evans, Science 240: 889-895 (1988)). Hence, a receptorpolypeptide dimer binds to a “whole-site,” with each receptorpolypeptide individually binding to a “half-site.” Furthermore, these“half-sites” may be oriented in either a direct repeat, inverted repeator palindromic fashion. The EcR and USP native receptor polypeptidesrecognize a palindromic response element, unlike most class II receptorproteins which recognize direct repeat response elements withappropriate spacing.

Additional flexibility in controlling gene expression by the presentinvention may be obtained by using DNA binding domains and responseelements from other transcriptional activators, which include but is notlimited to the LexA or GAL4 proteins. The DNA binding domain from theLexA protein encoded by the lexA gene from E. coli and its complementarybinding site (Brent and Ptashne, Cell 43:729-736, (1985), whichdescribes a LexA/GAL4 transcriptional activator) can be utilized. TheGAL4 protein of yeast (Sadowski et al. Nature 335: 563-564 (1988), whichdescribes a GAL4-VP16 transcriptional activator). In one preferredembodiment of the invention, a chimeric receptor polypeptide isconstructed by fusing the GAL4 DNA binding domain to a moiety containingthe ligand binding domain from EcR which, when heterodimerized with USPor a USP-moiety, can control expression of a target polypeptide.

Yet a further degree of flexibility in controlling gene expression canbe obtained by combining response elements which form complementarybinding pairs with DNA binding domains from different types oftranscriptional activators, i.e., using overlapping response elementsfrom GAL4 and a member of the steroid and thyroid hormone superfamily ofnuclear receptors. One example is the 5′ regulatory region of a targetexpression cassette which comprises the response element from GAL4 andoverlaps with the response element of T₃R. When T₃R proteinshomodimerize in the absence of chemical ligand, they will recognizetheir own response element and bind to it, thereby blocking the adjacentresponse element for GAL4. There is no activation of the 5′ regulatoryregion of the target expression cassette in this situation. Uponaddition of a complementary ligand for T₃R, the homodimer is separatedand released from its response element, unmasking the adjacent responseelement for GAL4. Once unmasked, the chimeric receptor polypeptidesutilizing the DNA binding domain from GAL4 may bind to the responseelement under appropriate dimerization conditions and thereby activateexpression of the target polypeptide.

The Transactivation Domain

Transactivation domains can be defined as amino acid sequences that,when combined with the DNA binding domain in a receptor polypeptide,increase productive transcription initiation by RNA polymerases. (Seegenerally Ptashne, Nature 335: 683-689 (1988)). Differenttransactivation domains are known to have different degrees ofeffectiveness in their ability to increase transcription inititiation.In the present invention it is desirable to use transactivation domainswhich have superior transactivating effectiveness in plant cells inorder to create a high level of target polypeptide expression inresponse to the presence of chemical ligand. Transactivation domainswhich have been shown to be particularly effective in the method of thepresent invention include but are not limited to VP16 (isolated from theherpes simplex virus) and C1 (isolated from maize). In one preferredembodiment of the present invention, the transactivation domain fromVP16 is fused to a USP-moiety for use as one monomer of a receptorpolypeptide heterodimer for controlling target polypeptide expression inplants. A further preferred embodiment is the fusion of thetransactivation domain from C1 to a EcR-moiety as a monomer. Othertransactivation domains may also be effective.

Repression of Gene Expression

As described above, the method of the present invention can be used toincrease gene expression over a minimal, basal level. One of theoutstanding benefits of the present method, however, is that it can alsobe used for decreasing or inhibiting gene expression, i.e., generepression. Repression can be achieved by the formation of homodimerswhere the half-sites of the response element have an linear orientationdistinct from the linear orientation which permits heterodimer binding.Under these conditions, homodimers bound to the 5′ regulatory region ofthe target expression cassette repress gene expression since theyinterfere with the transcription process. Hence, gene repression can beaccomplished by inclusion in the 5′ regulatory region an responseelement or response elements which permit homodimer binding. In oneembodiment of the invention, gene repression is achieved by binding of ahomodimer of USP or EcR to the 5′ regulatory region of a targetexpression cassette which comprises a complementary direct repeathalf-site.

Gene repression caused by homodimer binding would be released byaddition of a chemical ligand which triggers heterodimerization. Thisheterodimerization then activates the 5′ regulatory region of the targetexpression cassette. For example, in a transgenic plant expressing USPand EcR receptor polypeptides and comprising a target expressioncassette having both a palindrome half site response element and adirect repeat (or inverted repeat) half site response elementcomplementary to the DNA binding domain of USP and EcR, the expressionof the target polypeptide will be repressed. In the presence of RH 5992,or other chemical ligand which binds to the ligand binding domain of EcRor USP, or both, heterodimerization with USP and consequent binding tothe other response element present occurs, which in turn leads toactivation of the 5′ regulatory region of the target expressioncassette. Thus, repression of the target expression cassette would bereleased.

Controlling Gene Expression in Plants

For expression in plants, suitable promoters must be chosen for both thereceptor expression cassettes and the target expression cassette. Unlessspecifically noted, the promoters discussed below may be used to directexpression in plants of either the receptor polypeptides or the targetpolypeptide. These promoters include, but are not limited to,constitutive, inducible, temporally regulated, developmentallyregulated, chemically regulated, tissue-preferred and tissue-specificpromoters. Preferred constitutive promoters include but are not limitedto the CaMV 35S and 19S promoters (U.S. Pat. No. 5,352,605).Additionally preferred promoters include but are not limited to one ofseveral of the actin genes, which are known to be expressed in most celltypes. The promoter described by McElroy et al., Mol. Gen. Genet. 231:150-160 (1991), can be easily incorporated into the receptor expressioncassettes of the present invention and are particularly suitable for usein monocotyledonous hosts. Yet another preferred constitutive promoteris derived from ubiquitin, which is another gene product known toaccumulate in many cell types. The ubiquitin promoter has been clonedfrom several species for use in transgenic plants (e.g. sunflower—Binetet al., Plant Science 79: 87-94 (1991); maize—Christensen et al., PlantMolec. Biol. 12: 619-632 (1989)). The maize ubiquitin promoter has beendeveloped in transgenic monocot systems and its sequence and vectorsconstructed for transformation of monocotyledonous plants are disclosedin patent publication EP 0 342 926. The ubiquitin promoter is suitablefor use in the present invention in transgenic plants, especiallymonocotyledons. Further useful promoters are the U2 and U5 snRNApromoters from maize (Brown et al., Nucleic Acids Res. 17: 8991 (1989))and the promoter from alcohol dehydrogenase (Dennis et al., NucleicAcids Res. 12: 3983 (1984))

Tissue-specific or tissue-preferential promoters useful in the presentinvention in plants, particularly maize, are those which directexpression in root, pith, leaf or pollen. Such promoters are disclosedin U.S. Pat. No. 5,625,136, herein incorporated by reference in itsentirety. Also useful are promoters which confer seed-specificexpression, such as those disclosed by Schernthaner et al, EMBO J. 7:1249 (1988); anther-specific promoters ant32 and ant43D disclosed inU.S. Pat. No. 5,477,002, herein incorporated by reference in itsentirety; anther (tapetal) specific promoter B6 (Huffman et al., J.Cell. Biochem. 17B: Abstract #D209 (1993)); pistil-specific promoterssuch as a modified S13 promoter (Dzelkalns et al., Plant Cell 5:855(1993)).

Also useful in the present invention are chemically-induced promoters.Particular promoters in this category useful for directing theexpression of the receptor polypeptides or target polypeptide in plantsare disclosed, for example, in U.S. Pat. No. 5,614,395, hereinincorporated by reference in its entirety.

The 5′ regulatory region of either the receptor expression cassette orthe target expression cassette may also include other enhancingsequences. Numerous sequences have been found to enhance gene expressionin transgenic plants. For example, a number of non-translated leadersequences derived from viruses are known to enhance expression.Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the“Ω-sequence”), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa MosaicVirus (AMV) have been shown to be effective in enhancing expression(e.g. Gallie et al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski etal. Plant Molec. Biol. 15: 65-79 (1990)). Other leaders known in the artinclude but are not limited to:

Picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. PNAS USA86:6126-6130 (1989));

Potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allisonet al., (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology,154:9-20);

Human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejak,D. G., and Sarnow, P., Nature, 353: 90-94 (1991);

Untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4), (Jobling, S. A., and Gehrke, L., Nature, 325:622-625(1987);

Tobacco mosaic virus leader (TMV), (Gallie, D. R. et al., MolecularBiology of RNA, pages 237-256 (1989); and

Maize Chlorotic Mottle Virus leader (MCMV) (Lommel, S. A. et al.,Virology, 81:382-385 (1991). See also, Della-Cioppa et al., PlantPhysiology, 84:965-968 (1987).

Various intron sequences have been shown to enhance expression whenadded to the 5′ regulatory region, particularly in monocotyledonouscells. For example, the introns of the maize Adh1 gene have been foundto significantly enhance the expression of the wild-type gene under itscognate promoter when introduced into maize cells (Callis et al., GenesDevelep 1:1183-1200 (1987)).

In addition to promoters, a variety of 3′ transcriptional terminatorsare also available for use in the present invention. Transcriptionalterminators are responsible for the termination of transcription andcorrect mRNA polyadenylation. Appropriate transcriptional terminatorsand those which are known to function in plants include the CaMV 35Sterminator, the tml terminator, the nopaline synthase terminator, thepea rbcS E9 terminator and others known in the art. These can be used inboth monocotyledons and dicotyledons.

In addition to incorporating one or more of the aforementioned elementsinto the 5′ regulatory regron of a target expression cassette, otherelements peculiar to the target expression cassette may also beincorporated. Such elements include but are not limited to a minimalpromoter. By minimal promoter it is intended that the basal promoterelements are. inactive or nearly so without upstream activation. Such apromoter has low background activity in plants when there is notransactivator present or when enhancer or response element bindingsites are absent. One minimal promoter that is particularly useful fortarget genes in plants is the Bz1 minimal promoter which is obtainedfrom the bronzel gene of maize. The Bz1 core promoter was obtained fromthe “myc” mutant Bz1-luciferase construct pBz1LucR98 via cleavage at theNheI site located at −53 to −58. Roth et al., Plant Cell 3: 317 (1991).The derived Bz1 core promoter fragment thus extends from −53 to +227 andincludes the Bz1 intron-1 in the 5′ untranslated region.

Plant Transformation

The expression cassettes of the present invention can be introduced intothe plant cell in a number of art-recognized ways. Those skilled in theart will appreciate that the choice of method might depend on the typeof plant, i.e. monocotyledonous or dicotyledonous, targeted fortransformation. Suitable methods of transforming plant cells include,but are not limited to, microinjection (Crossway et al., BioTechniques4:320-334 (1986)), electroporation (Riggs et al., Proc. Natl. Acad. Sci.USA 83:5602-5606 (1986), Agrobacterium-mediated transformation (Hincheeet al., Biotechnology 6:915-921 (1988)), direct gene transfer(Paszkowski et al., EMBO J. 3:2717-2722 (1984)), and ballistic particleacceleration using devices available from Agracetus, Inc., Madison, Wis.and BioRad, Hercules, Calif. (see, for example, Sanford et al., U.S.Pat. No. 4,945,050; and McCabe et al., Biotechnology 6:923-926 (1988)).Also see, Weissinger et al., Annual Rev. Genet. 22:421-477 (1988);Sanford et al., Particulate Science and Technology 5:27-37(1987)(onion); Christou et al., Plant Physiol. 87:671-674(1988)(soybean); McCabe et al., Bio/Technology 6:923-926(1988)(soybean); Datta et al., Bio/Technology 8:736-740 (1990)(rice);Klein et al., Proc. Natl. Acad. Sci. USA, 85:4305-4309 (1988)(maize);Klein et al., Bio/Technology 6:559-563 (1988)(maize); Klein et al.,Plant Physiol. 91:440-444 (1988)(maize); Fromm et al., Bio/Technology8:833-839 (1990)(maize); and Gordon-Kamm et al., Plant Cell 2:603-618(1990)(maize); Svab et al., Proc. Natl. Acad. Sci. USA 87: 8526-8530(1990)(tobacco chloroplast); Koziel et al., Biotechnology 11: 194-200(1993)(maize); Shimamoto et al., Nature 338: 274-277 (1989)(rice);Christou et al., Biotechnology 9: 957-962 (1991)(rice); European PatentApplication EP 0 332 581 (orchardgrass and other Pooideae); Vasil etal., Biotechnology 11: 1553-1558 (1993)(wheat); Weeks et al., PlantPhysiol. 102: 1077-1084 (1993)(wheat).

One particularly preferred set of embodiments for the introduction ofthe expression cassettes of the present invention into maize bymicroprojectile bombardment is described in U.S. Ser. No. 08/008,374,herein incorporated by reference in its entirety. An additionalpreferred embodiment is the protoplast transformation method for maizeas disclosed in European Patent Serial No. 08/008,374, hereinincorporated by reference in its entirety. An additional preferredembodiment is the protoplast transformation method for maize asdisclosed in European Patent Application EP 0 292 435, as well as inU.S. Pat. No. 5,350,689, hereby incorporated by reference in itsentirety. One particularly preferred set of embodiments for theintroduction of the expression cassettes of the present invention intowheat by microprojectile bombardment can be found in U.S. Ser. No.08/147,161, herein incorporated by reference in its entirety.

Transformation of plants can be undertaken with a single DNA molecule ormultiple DNA molecules (i.e. co-transformation), and both thesetechniques are suitable for use with the expression cassettes of thepresent invention. Numerous transformation vectors are available forplant transformation, and the expression cassettes of this invention canbe used in conjunction with any such vectors. The selection of vectorwill depend upon the preferred transformation technique and the targetspecies for transformation.

Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). Inone preferred embodiment, the expression cassettes of the presentinvention may be inserted into either of the binary vectors pCIB200 andpCIB2001 for use with Agrobacterium. These vector cassettes forAgrobacterium-mediated transformation wear constructed in the followingmanner. pTJS75kan was created by NarI digestion of pTJS75 (Schmidhauser& Helinski, J Bacteriol. 164: 446-455 (1985)) allowing excision of thetetracycline-resistance gene, followed by insertion of an AccI fragmentfrom pUC4K carrying an NPTII (Messing & Vierra, Gene 19: 259-268 (1982);Bevan et al., Nature 304: 184-187 (1983); McBride et al., PlantMolecular Biology 14: 266-276 (1990)). XhoI linkers were ligated to theEcoRV fragment of pCIB7 which contains the left and right T-DNA borders,a plant selectable nos/nptII chimeric gene and the pUC polylinker(Rothstein et al., Gene 53: 153-161 (1987)), and the XhoI-digestedfragment was cloned into SalI-digested pTJS75kan to create pCIB200 (seealso EP 0 332 104, example 19). pCIB200 contains the following uniquepolylinker restriction sites: EcoRI, SstI, KpnI, BglII, XbaI, and SaII.The plasmid pCIB2001 is a derivative of pCIB200 which was created by theinsertion into the polylinker of additional restriction sites. Uniquerestriction sites in the polylinker of pCIB2001 are EcoRI, SstI, KpnI,BglII, XbaI, SalI, MluI, BclI, AvrII, ApaI, HpaI, and StuI. pCIB2001, inaddition to containing these unique restriction sites also has plant andbacterial kanamycin selection, left and right T-DNA borders forAgrobacterium-mediated transformation, the RK2-derived trfA function formobilization between E. coli and other hosts, and the OriT and OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for thecloning of plant expression cassettes containing their own regulatorysignals.

An additional vector useful for Agrobacterium-mediated transformation isthe binary vector pCIB 10, which contains a gene encoding kanamycinresistance for selection in plants, T-DNA right and left bordersequences and incorporates sequences from the wide host-range plasmidpRK252 allowing it to replicate in both E. coli and Agrobacterium. Itsconstruction is described by Rothstein et al., Gene 53: 153-161 (1987).Various derivatives of pCIB10 have been constructed which incorporatethe gene for hygromycin B phosphotransferase described by Gritz et al.,Gene 25: 179-188 (1983). These derivatives enable selection oftransgenic plant cells on hygromycin only (pCIB743), or hygromycin andkanamycin (pCIB715, pCIB717).

Methods using either a form of direct gene transfer orAgrobacterium-mediated transfer usually, but not necessarily, areundertaken with a selectable marker which may provide resistance to anantibiotic (e.g., kanamycin, hygromycin or methotrexate) or a herbicide(e.g., phosphinothricin). The choice of selectable marker for planttransformation is not, however, critical to the invention.

For certain plant species, different antibiotic or herbicide selectionmarkers may be preferred. Selection markers used routinely intransformation include the nptII gene which confers resistance tokanamycin and related antibiotics (Messing & Vierra, Gene 19: 259-268(1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene whichconfers resistance to the herbicide phosphinothricin (White et al., NuclAcids Res 18: 1062 (1990), Spencer et al., Theor Appl Genet 79:625-631(1990)), the hph gene which confers resistance to the antibiotichygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931), andthe dhfr gene, which confers resistance to methotrexate (Bourouis etal., EMBO J. 2: 1099-1104 (1983)).

One such vector useful for direct gene transfer techniques incombination with selection by the herbicide Basta (or phosphinothricin)is pCIB3064. This vector is based on the plasmid pCIB246, whichcomprises the CaMV 35S promoter in operational fusion to the E. coli GUSgene and the CaMV 35S transcriptional terminator and is described in thePCT published application WO 93/07278, herein incorporated by reference.One gene useful for conferring resistance to phosphinothricin is the bargene from Streptomyces viridochromogenes (Thompson et al., EMBO J 6:2519-2523 (1987)). This vector is suitable for the cloning of plantexpression cassettes containing their own regulatory signals.

An additional transformation vector is pSOG35 which utilizes the E. coligene dihydrofolate reductase (DHFR) as a selectable marker conferringresistance to methotrexate. PCR was used to amplify the 35S promoter(˜800 bp), intron 6 from the maize Adh1 gene (˜550 bp) and 18 bp of theGUS untranslated leader sequence from pSOG10. A 250 bp fragment encodingthe E. coli dihydrofolate reductase type II gene was also amplified byPCR and these two PCR fragments were assembled with a SacI-PstI fragmentfrom pBI221 (Clontech) which comprised the pUC19 vector backbone and thenopaline synthase terminator. Assembly of these fragments generatedpSOG19 which contains the 35S promoter in fusion with the intron 6sequence, the GUS leader, the DHFR gene and the nopaline synthaseterminator. Replacement of the GUS leader in pSOG19 with the leadersequence from Maize Chlorotic Mottle Virus check (MCMV) generated thevector pSOG35. pSOG19 and pSOG35 carry the pUC-derived gene forampicillin resistance and have HindIII, SphI, PstI and EcoRI sitesavailable for the cloning of foreign sequences.

Control of Plant Fertility

One of the advantageous aspects of the present invention is its use inthe control of effective fertilization in plants under field conditions.Effective fertilization is the formation of viable zygotes. Plantfertility can be controlled by incorporating a nucleotide sequenceencoding an appropriate target polypeptide into the target expressioncassette wherein the expression of the target polypeptide which willrender the fertilization process ineffective, meaning that the formationof viable zygotes will be prevented. Ineffective fertilization may becaused by a variety of means. These include but are not limited to, 1)disruption or alteration of those processes which are critical toformation of viable gametes, 2) pollen or ovules that, if formed, arenot functional, or 3) failure of the embryo sac, pistil, stigma ortransmitting tract to develop properly. In the present invention, achemical ligand is applied to transgenic plants under field conditions,wherein the expression of a target polypeptide is activated, wherebyfertilization is rendered ineffective.

Useful coding sequences for the target polypeptide include but are notlimited to any sequence which encodes a product capable of renderingfertilization ineffective. These coding sequences can be of either ahomologous or heterologous origin. The gene products of those codingsequences include, but are not limited to:

Diphtheria Toxin A-chain (DTA), which inhibits protein synthesis,Greenfield et al., Proc. Natl. Acad., Sci.:USA, 80:6853 (1983); Palmiteret al., Cell, 50:435 (1987);

Pectate lyase pelE from Erwinia chrysanthemi EC16, which degradespectin, causing cell lysis. Keen et al., J. Bacteriology, 168:595(1986);

T-urf13 (TURF-13) from cms-T maize mitochondrial genomes; this geneencodes a polypeptide designated URF13 which disrupts mitochondrial orplasma membranes. Braun et al., Plant Cell, 2:153 (1990); Dewey et al.,Proc. Natl. Acad. Sci.:USA, 84:5374 (1987); Dewey et al., Cell, 44:439(1986);

Gin recombinase from phage Mu a gene, which encodes a site-specific DNArecombinase which will cause genome rearrangements and loss of cellviability when expressed in cells of plants. Maeser et al., Mol. Gen.Genet., 230:170-176 (1991);

Indole acetic acid-lysine synthetase (iaaL) from Pseudomonas syringae,which encodes an enzyme that conjugates lysine to indoleacetic acid(IAA). When expressed in the cells of plants, it causes altereddevelopments due to the removal of IAA from the cell via conjugation.

Romano et al., Genes and Development, 5:438-446 (1991); Spena et al.,Mol. Gen. Genet., 227:205-212 (1991); Roberto et al., Proc. Natl. Acad.Sci.:USA, 87:5795-5801;

Ribonuclease from Bacillus amyloliquefaciens, also known as barnase,digests mRNA in those cells in which it is expressed, leading to celldeath. Mariani et al., Nature 347: 737-741 (1990); Marian et al., Nature357: 384-387 (1992); and,

CytA toxin gene from Bacillus thuringiensis israeliensis which encodes aprotein that is mosquitocidal and hemolytic. When expressed in plantcells, it causes death of the cell due to disruption of the cellmembrane. McLean et al., J. Bacteriology, 169:1017-1023 (1987); Ellar etal., U.S. Pat. No. 4,918,006 (1990).

Such polypeptides also include Adenine Phosphoribosyltransferase (APRT)Moffatt and Somerville, Plant Physiol., 86:1150-1154 (1988); DNAse,RNAse; protease; salicylate hydroxylase; etc.

It is further recognized that the target expression cassette maycomprise a 5′ regulatory region operably linked to a nucleotide sequencewhich, when transcribed, produces an antisense version of a codingsequence critical to the formation of viable gametes, such as APRT.Alternately, ribozymes can be utilized which target mRNA from a genewhich is critical to gamete formation or function. Such ribozymes willcomprise a hybridizing region of about nine nucleotides which iscomplementary in nucleotide sequence to at least part of the target RNAand a catalytic region which is adapted to cleave the target RNA.Ribozymes are described in EPA No. 0 321 201 and WO88/04300 hereinincorporated by reference. See, also Haseloff and Gerlach, Nature,334:585-591 (1988); Fedor and Uhlenbeck Proc. Natl. Acad. Sci.: USA,87:1668-1672 (1990); Cech and Bass, Ann. Rev. Biochem., 55:599-629(1986); Cech, T. R., 236:1532-1539 (1987); Cech, T. R. Gene, 73:259-271(1988); and, Zang and Cech, Science, 231:470-475 (1986).

It is recognized that the above nucleotide sequences encoding a targetpolypeptide can also be operably linked to a 5′ regulatory sequencewhich directs its expression in a tissue- or cell-specific manner. Themeans to provide such tissue- or cell-specific expression has beendescribedabove. This specificity in expression ensures that the effectof the target polypeptide will be exerted only on those tissues or cellswhich are necessary for the formation of viable gametes and will not bedeleterious to the plant beyond its affect on fertility.

It is recognized as within the scope of the invention that either malefertility of the transgenic plants, female fertility of the transgenicplants, or both, may be controlled. Male sterility is the failure orinability to produce functional or viable pollen. Male sterility mayresult from defects leading to the non-formation of pollen or to thelack of functional ability in the pollen when it is formed. Therefore,either pollen is not formed or, if formed, it is either non-viable orincapable of effective fertilization under normal conditions.

Female sterility is the failure or inability to produce functional orviable megaspores or embryo sacs, or other tissues required for pollengermination, growth or fertilization. Female sterility may result fromdefects leading to the non-formation of the megaspores or embryo sac, orfailure of the ovary, ovule, pistil, stigma, or transmitting tract todevelop properly. Therefore, either a viable embryo sac fails todevelop, or if formed, it is incapable of effective fertilization undernormal conditions.

For example, a transgenic plant can be obtained which expresses the EcRand USP receptor polypeptides in anthers using an anther-specificpromoter fused to the appropriate nucleotide sequences. In addition, thetransgenic plant will further comprise a target expression cassettehaving a 5 ′ regulatory sequence comprising the appropriate responseelement sequence with the core promoter elements from Bz1, operablylinked to the coding sequence for the ribonuclease barnase. Uponapplication of RH 5992 as chemical ligand to the transgenic plant,heterodimerization of the EcR and USP receptor polypeptides occurs,activating the 5′ regulatory sequence of the target expression cassetteand with subsequent production of the target of polypeptide bamiase. Theresulting expression of barnase specifically in the anthers causes celldeath and consequent male sterility. A similar combination of receptorpolypeptides and target expression cassette, using a pistil-specificpromoter operably linked to the nucleotide sequences encoding thereceptor polypeptides, can produce female sterility.

Alternatively, the plant could be engineered wherein expression of thetarget polypeptide restores fertility to a male-sterile orfemale-sterile plant. For example, a plant could be obtained thatexpressed the bamase gene under control of the Ant43D, Ant32 or B6promoters, or as described in Mariani et al., Nature 347: 737-741 (1990)and Mariani et al., Nature 357: 384-387 (1992), under control of theTA29 promoter. These plants would additionally comprise the receptorexpression cassettes which express the EcR and USP receptor polypeptidefrom either the same anther-specific promoter or from a constitutivepromoter such as maize ubiquitin, 35S or rice actin. These plants wouldfurther comprise a target expression cassette having a S regulatorysequence comprising the appropriate response element sequence with thecore promoter elements from Bz1, operably linked to the coding sequencefor the barnase inhibitor barstar. The plants would be male-sterile, butupon application of RH5992 as a chemical ligand, heterodimerization ofUSP and EcR receptor polypeptides would occur, resulting in activationof the 5′ regulatory sequence of the target expression cassette andproduction of the target polypeptide barstar. Barstar would inhibit theribonuclease activity of the barnase polypeptide, and anther and pollendevelopment would proceed normally. Fertility would be restored.

A similar approach could be used to control female sterility. Byutilizing promoters specific for expression in the female reproductivetissues to drive barnase expression instead of the anther-specificpromoters, female-sterile plants would be obtained. Induction bychemical ligand of the target expression cassette comprising the barstarcoding sequence would result in restoration of female fertility.

The above approaches could utilize any female- or male-sterility genefor which a restorer gene could be devised. Other potential restorergenes are described in European Patent Application EP 412 911.

Therefore, the present invention can be used in any plant which can betransformed and regenerated to obtain transgenic plants in which maleand/or female sterility can be controlled by the application of theappropriate chemical ligand. The control of plant fertility isparticularly useful for the production of hybrid seed. In order toproduce hybrid seed uncontaminated with trio selfed seed, pollinationcontrol methods must be implemented to ensure cross-pollination and notself-pollination. This is usually accomplished by mechanical, genetic orchemical hybridizing agents (CHAs). For example, in maize the currentpractice is mechanical detasseling of the female (or seed) parent, whichis a time consuming and labor intensive process. In wheat, controllingfertility by mechanical means is impractical on a seed production scale,and genetic sources of control are not established. The use of thepresent invention in the production of hybrid seed offers the advantagesof reliability, ease of use and control of either male or femalefertility.

The transgenic plants containing the appropriate receptor expressioncassettes and target expression cassette can be made homozygous andmaintained indefinitely. To obtain hybrid seed, homozygous lines ofParent 1 and Parent 2 are crossed. In one example of using the presentinvention to produce hybrid seed, Parent 1 is engineered to be malesterile in the presence of the appropriate chemical ligand whereasParent 2 is engineered to be female sterile in the presence of anappropriate chemical ligand. After application of an appropriatechemical ligand, which is determined by the choice of ligand bindingdomain present in the receptor polypeptides, the only successful seedproduction will be a result of Parent 2 pollen fertilizing Parent 1ovules. In a second example of using the present invention, Parent 1 isengineered to be male-sterile in the absence of the appropriate chemicalligand and Parent 2 is engineered to be female sterile in the absence ofthe chemical ligand. The appropriate chemical is applied to maintaineach line through self-fertilization. To produce hybrid seed, the twoparent lines are interplanted, and only hybrid seed is obtained.Fertility is restored to the progeny hybrid plants by an introducedrestorer gene. By these means any desired hybrid seed may be produced.

EXAMPLES

The following examples further describe the materials and methods usedin carrying out the invention and the subsequent results. They areoffered by way of illustration, and their recitation should not beconsidered as a limitation of the claimed invention.

Example 1 Construction of a Plant-expressible Receptor ExpressionCassette Encoding the Ecdysone Receptor

The DNA coding region for the Ecdysone Receptor (EcR) of Drosophila wasisolated from a cDNA library derived from Canton S pupae (day 6)prepared in λgtl1 (Clontech, cat. no. IL 1005b), and from fragmentsgenerated by genomic PCR with oligonucleotides designed from thepublished sequence of the B1 isoform of the EcR (Koelle et al., Cell67:59, 1991). The B1 isoform EcR sequence was confirmed by automatedsequence analysis using standard methods and alignment with thepublished sequence (Talbot et al., Cell 73:1323, 1993). The expressedfull length EcR coding region was modified to contain a BamHI siteimmediately upstream from the start codon using the oligonucleotide SF43(5′-CGC GGA TCC TAA ACA ATG AAG CGG CGC TGG TCG AAC AAC GGC-3′; SEQ IDNO:1) in a PCR reaction. The plant expression vectors pMF6 and pMF7contain a Cauliflower Mosaic Virus 35S promoter (CaMV 35S), a maize Adh1Intron1, and a nopaline synthetase polyadenylation and terminationsignal (See Goff et al., Genes and Development 5:298, 1991). The vectorspMF6 and pMF7 differ only in the orientation of the polylinker used forinsertion of the desired coding sequence. The full length EcR codingsequence was ligated into the plant CaMV 35S expression vector pMF6 byusing the flanking BamHI restriction sites. This receptor expressioncassette is referred to as 35S/EcR.

Example 2 Construction of a Plant-expressible Receptor ExpressionCassette Encoding the Ultraspiracle Receptor

The cDNA encoding the native Ultraspiracle receptor (USP) of Drosophilais described by Henrich et al., Nucleic Acids Research 18:4143 (1990).The full length USP coding sequence with the flanking 5′ and 3′untranslated regions was ligated into the plant expression vector pMF7(described in Example 1) using the flanking EcoRI restriction sites.This receptor expression cassette is referred to as 35S/USP.

Example 3 Construction of a Receptor Expression Cassette having the DNABinding Domain from GAL4 and the Ligand Binding Domain from EcR

A receptor expression cassette was constructed where the DNA bindingdomain of EcR is replaced by the DNA binding domain of GAL4 fused at theN-terminal position. The DNA coding region for the EcR of Drosophila wasobtained as described in Example 1. The coding sequence for the DNAbinding domain of GAL4 was subcloned from plasmid pMA210. Ma andPtashne, Cell, 48: 847 (1987).

A receptor expression cassette encoding a GAL4-EcR chimeric receptorpolypeptide was constructed by fusion of the DNA binding domain of GAL4to the ligand binding domain and carboxy terminus of EcR. To make thefusion, the oligonucleotide SF23 (5′-CGC GGG ATC CAT GCG GCC GGA ATG CGTCGT CCC G-3′; SEQ ID NO:2) was used to introduce by PCR a BamHI siteinto the cDNA sequence for EcR at the nucleotide position equivalent toamino acid residue 330 (immediately following the EcR DNA-bindingdomain). The resulting truncated EcR coding sequence (EcR³³¹⁻⁸⁷⁸) wassubcloned into the plasmid pKS+ (Stratagene).

A subclone of GAL4 was obtained from plasmid pMA210 which contained thecoding sequence of the DNA binding domain (amino acids 1-147) bysubcloning the amino terminus of GAL4 to the ClaI site into pSK+(Stratagene) as previously described (Goff et al., Genes and Development5:298, 1991). This plasmid was designated pSKGAL2, and was cut with ClaIand KpnI and the following double stranded oligonucleotide was inserted:

5′-CGGGGGATCCTAAGTAAGTAAGGTAC-3′ (SEQ ID NO:10)3′-CCCCTAGGATTCATTCATTC-5′ (SEQ ID NO:11)The resulting plasmid was designated pSKGAL2.3. The complete fusion35S/GAL4-EcR³³⁰⁻⁸⁷⁸ was generated using the BamHI sites in thepolylinkers flanking the DNA binding domain of GAL4 in pSK+ and theEcR³³⁰⁻⁸⁷⁸ moiety in pKS+. These coding sequences were ligated into themonocot expression vector pMF6 (described in Example 1) via the use ofthe flanking EcoRI restriction sites. This receptor expression cassetteis referred to as 35S/GAL4-EcR³³⁰⁻⁸⁷⁸.

Example 4 Construction of a Plant-expressible Receptor ExpressionCassette having the Ligand Binding Domain from Ultraspiracle and theTransactivation Domain from VP16

A receptor expression cassette was constructed which comprises theligand binding domain of USP with the transactivation domain of VP16fused to either the N-terminus or C-terminus of the USP polypeptide.

To construct the receptor expression cassette encoding a chimericpolypeptide having the transactivation domain of VP16 at the C-terminalposition, the carboxy-terminus and stop codon of the cDNA for thereceptor USP (described in Example 2) were removed by subcloning intopKS+ (Stratagene) using the XhoI site at USP nucleotide number 1471 ofthe coding sequence. The resulting USP subclone encoding amino acids 1to 490 was fused to the transactivation domain of VP16 using theflanking KpnI restriction site of the USP subclone, and the KpnI site ofpSJTl 193CRF3 which encodes the carboxy-terminal 80 amino acids of VP16(Triezenberg et al., Genes and Develop. 2: 718-729 (1988)). Theresulting USP-VP16 fusion was cloned into the CaMV 35S plant expressionvector pMF7 (described in Example 1) using the EcoRI and BamHIrestriction enzyme sites flanking the coding sequence of USP-VP16. Thisreceptor expression cassette is referred to as 35S/USP-VP16.

The USP derivative with the transcriptional activation domain fused tothe amino-terminus was constructed by first engineering a BamHI siteadjacent to the USP start codon using the oligonucleotide SF42 (5′-CGCGGA TCC ATG GAC AAC TGC GAC CAG GAC-3′; SEQ ID NO:3) in a PCR reaction.The stop codon in VP16 was eliminated and a flanking BamHI siteintroduced using the oligonucleotide SF37 (5′-GCG GGA TCC CCC ACC GTACTC GTC AAT TC-3′; SEQ ID NO:4) and a start codon with a plant consensussequence as well as a BamHI site was introduced at the amino terminalend using the oligonucleotide SAl15 (5′-GTC GAG CTC TCG GAT CCT AAA ACAATG GCC CCC CCG ACC GAT GTC-3′; SEQ ID NO:5) as primers in a PCRreaction. The resulting VP16 activation domain and USP coding sequence(encoding amino acids 1 to 507) were joined in frame by the adjacentBamHI sites, and the VP16-USP coding sequence was inserted into the CAMV35S plant expression vector pMF7 by the 5′ BamHI and 3′ EcoRI sites.This receptor expression cassette is referred to as 35VP16-USP.

Example 5 Construction of a Receptor Expression Cassette having the DNABinding Domain and Ligand Binding Domain from EcR and theTransactivation Domain from the C1 Regulatory Gene of Maize

The EcR²²⁷⁻⁸²⁵-C1 fusion was generated by placing a start codonimmediately before the EcR DNA binding domain with the oligonucleotideSF30 (5′-CGC-GGA-TCC-ATG-GGT-CGC-GAT-GAT-CTC-TCG-CCT-TC-3′; SEQ ID NO:8)used in a PCR reaction on the full length EcR coding sequence. Thecoding sequence for the transcriptional activation domain (amino acids219-273) of the maize C1 protein (Goff et al. Genes and Develop. 5:298-309 (1991)) was fused in frame to the coding sequence for aminoacids 51 to 825 of EcR (at the EcR KpnI restriction enzyme site). The C1transactivation domain was linked to EcR by a polylinker encodingVPGPPSRSRVSISLHA (SEQ ID NO:9). The 35S/EcR²²⁷⁻⁸²⁵-C1 plant expressionvector fusion was constructed by insertion of a BamHI fragment carryingthe coding sequence into the pMF7 vector. This receptor expressioncassette is referred to as 35S/EcR²²⁷⁻⁸²⁵-C1.

Example 6 Construction of a Receptor Expression Cassette having the DNABinding Domain from GAL4, the Ligand Binding Domain from EcR and theTransactivation Domain from the C1 Regulatory Gene of Maize

A GAL4-EcR³³⁰⁻⁸²⁵-C1 fusion was constructed using the GAL4-EcR³³⁰⁻⁸⁷⁸construct described in Example 3 and the EcR²²⁷⁻⁸²⁵-C1 construct ofExample 5. The sequence of the EcR coding region (at amino acid 456) wasexchanged at the AatII site. This receptor expression cassette isreferred to as 35S/GAL4-EcR³³⁰⁻⁸²⁵-C1.

Example 7 Construction of a Plant-expressible Target Expression CassetteEncoding Firefly Luciferase having the Response Element for the GAL4 DNABinding Domain

The plant-expressible target expression cassette encoding fireflyluciferase having the response element for the DNA binding domain ofGAL4 was constructed in the following manner.

The maize Bronze-1 (Bz1) core promoter driving the synthesis of fireflyluciferase was removed from the Bz1 reporter pBz1LucR98 (Roth et al.,Plant Cell 3:317, 1991) via the NheI and SphI sites and placed in apUC6S-derived plasmid carrying the luciferase gene. The modified Bz1core promoter contains an NheI site (GCTAGC) and Bz1 promoter sequencesup to nucleotide position −53 (Roth et al., Plant Cell 3:317, 1991). TenGAL4 binding sites were removed from the GAL4 regulated reporterpGALLuc2 (Goff etal., Genes and Development 5:298, 1991) by digestionwith EcoRI and PstI and inserted into pBlueScript (Stratagene) using thesame restriction enzyme sites. The HindIII site at the 5′ end of theGAL4 binding sites was changed to a BamHI site by insertion of anHindIII/BamHI/HindIII adaptor, and the resulting BamHI fragmentcontaining the GAL4 binding sites was removed and placed into a BglIIsite upstream of the Bz1 core promoter driving luciferase. This targetexpression cassette is referred to as (GAL4_(b.s)))₁₀-Bz1_(TATA)/Luc.

Example 8 Construction of a Plant-expressible Target Expression CassetteEncoding Firefly Luciferase having the Response Element for EcR DNABinding Domain

The plant-expressible target expression cassette encoding fireflyluciferase having the response element for the DNA binding domain of EcRwas constructed in the following manner. The maize Bz1 corepromoter-luciferase construct in the pUC6S-derived plasmid as describedin Example 7 was used as the starting point. Double-stranded syntheticoligonucleotides containing the Drosophila hsp27 response element whichcomplements the DNA binding domain of EcR were constructed with BamHIand BglII cohesive ends (SF25: 5′-GAT CCG ACA AGG GTT CAA TGC ACT TGTCA-3′; SEQ ID NO:6) (SF26: 5′-GAT CTG ACA AGT GCA TTG AAC CCT TGT CG-3′;SEQ ID NO:7), phosphorylated, and ligated upstream of the Bz1 corepromoter by insertion into a unique BglII site. Multiple copies of theresponse element were obtained by sequential BglII digestion andinsertion of additional double-stranded oligonucleotides. This targetexpression cassette is referred to as either (EcRE)₅-Bz1/Luc,(EcRE)₆-Bz1/Luc, or (EcRE)₈-Bz1/Luc, depending on the number offull-site palindromic response elements contained within the promoterregion (5, 6, and 8, respectively).

Example 9 Transformation of Plant Cells and Control of TargetPolypeptide Expression by Receptor Polypeptides in the Presence of aChemical Ligand

Control of target polypeptide expression by various receptorpolypeptides, including the chimeric receptor polypeptides of thepresent invention, can be shown by simultaneously transforming plantcells with the necessary gene constructions using high velocitymicroprojectile bombardment, followed by biochemical assay for thepresence of the target polypeptide. The necessary gene constructionscomprise a first receptor expression cassette which encodes a firstreceptor polypeptide and a second receptor expression cassette whichencodes a second receptor polypeptide. In addition, a target expressioncassette which-encodes a target polypeptide is also necessary (FIG. 1).

The expression cassettes were simultaneously delivered to maizesuspension cells cultured in liquid N6 medium (Chu et al. ScientiaSinica XVIII:659-668, 1975) by high velocity microprojectile bombardmentusing standard techniques of DNA precipation onto microprojectiles andhigh velocity bombardment driven by compressed helium (PDS-1000/He,BioRad, Hercules, Calif.). Transfected cells were incubated in liquidsuspension in the presence of the appropriate chemical ligand forapproximately 48 hours in N6 media After incubation, the transformedcells were harvested then homogenized at 0° C. Debris in the extractswas removed by centrifugation at 10,000 g at 4° C. for 5 minutes. Targetpolypeptide expression was detected by assaying the extract for thepresence of the product encoded by the target expression cassette. Onecommonly used coding sequence for the target polypeptide when testingcontrol of expression by the receptor polypeptides in the presence of achemical ligand is firefly luciferase. The activity of fireflyluciferase is determined by quantitating the chemiluminescence producedby luciferase catalyzed phosphorylation of luciferin using ATP assubstrate (Promega Luciferase Kit, cat. no. E1500), using an AnalyticalLuminescence Model 2001 luminometer.

Example 10

The Receptor Polypeptides EcR and USP Activate Expression of a TargetPolypeptide in Plant Cells

Using the transformation method of Example 9, the receptor expressioncassette 35S/EcR (Example 1), the receptor expression cassette 35S/USP(Example 2) and the target expression cassette (EcRE)₅-Bz1/Luc (Example8) were co-transformed into maize cells (see FIG. 2). Transformed cellswere incubated in the presence of 10 μM RH5992 or 2 μM muristerone aschemical ligands for approximately 48 hours. Luciferase assays wereperfomed as described in Example 9. The results are presented in Table1.

TABLE 1 Presence of Chemical Luciferase Activity Receptor Ligand (lightunits) None None 427 35S/EcR None 295 35S/EcR + 35S/USP RH555992 86035S/EcR + 35S/USP Muristerone 1351 

The above results show that the 5′ regulatory region of the targetexpression cassette comprising the EcR response elements can beactivated in plant cells by the receptor polypeptides EcR and USP in thepresence of a complementary chemical ligand. The level of expression ofthe target polypeptide luciferase was about 2- to 3-fold above thatobserved in the absence of chemical ligand.

Example 11 The Receptor Polypeptides VP16-USP and EcR ActivateExpression of a Target Polypeptide in Plant Cells

Using the transformation method of Example 9, the receptor expressioncassette 35S/EcR (Example 1), the receptor expression cassette35S/VP16-USP (Example 4) and the target expression cassette(EcRE)₆-Bz1/Luc (Example 8) were co-transformed into maize cells.Transformed cells were incubated in the presence of 1 μM ecdysone, 10 μMRH5992 or 2 μM muristerone as chemical ligands for 48 hours. Luciferaseassays were performed as described in Example 9. The results arepresented in Table 2.

TABLE 2 Presence of Chemical Luciferase Activity Receptor Ligand (lightunits) None None 427 35S/EcR + 35S/VP16-USP None 4,486 35S/EcR +35S/VP16-USP ecdysone 7,420 35S/EcR + 35S/VP16-USP RH5992 7,00335S/EcR + 35S/VP16-USP Muristerone 12,374

The above results show that the 5′ regulatory region of the targetexpression cassette comprising the EcR response elements can beactivated in plant cells by the receptor polypeptide EcR and thechimeric receptor polypeptide VP16-USP in the presence of acomplementary chemical ligand. The level of expression of the targetpolypeptide luciferase was about 2- to 3-fold above that observed in theabsence of chemical ligand.

Example 12 The Receptor Polypeptides EcR²²⁷⁻⁸²⁵-C1 and USP ActivateExpression of a Target Polypeptide in Plant Cells

Using the transformation method of Example 9, the receptor expressioncassette 35S/USP (Example 2), the receptor expression cassette35S/EcR²²⁷⁻⁸²⁵-C1 (Example 5) and the target expression cassette(EcRE)₆-Bz1/Luc (Example 8) were co-transformed into maize cells.Transformed cells were incubated in the presence of 10 μM RH5992 aschemical ligand for 48 hours. Luciferase assays were performed asdescribed in Example 9. The results are presented in Table 3.

TABLE 3 Presence of Chemical Luciferase Activity Receptor Ligand (lightunits) None None  5,626 35S/USP + 35S/ None 10,024 EcR²²⁷⁻⁸²⁵-C135S/USP + 35S/ RH5992 24,631 EcR²²⁷⁻⁸²⁵-C1

The above results show that the 5′ regulatory region of the targetexpression cassette comprising the EcR response elements can beactivated in plant cells by the receptor polypeptide USP and thechimeric receptor polypeptide EcR²²⁷⁻⁸²⁵-C1 in the presence of acomplementary chemical ligand. The chimeric receptor polypeptide usesthe transactivation domain of the maize regulatory gene C1 fused to theC-termninus of a truncated EcR, where the truncation has removed thetransactivation domain of EcR. The level of expression of the targetpolypeptide luciferase was more than 2-fold above that observed in theabsence of chemical ligand.

Example 13 Activation of a Target Expression Cassette in Plant Cells isEnhanced by Using a Chimeric Receptor Polypeptide having a StrongTransactivation Domain

Using the transformation method of Example 9, the receptor expressioncassette 35S/GAL4-EcR³³⁰⁻⁸⁷⁸ (Example 3), the receptor expressioncassette 35S/USP-VP16 (Example 4) and the target expression cassette(GAL4)₁₀-Bz1/Luc (Example 7) were co-transformed into maize cells (FIG.3). Transformed cells were incubated in the presence of 10 μM RH5992 aschemical ligand for approximately 48 hours. Luciferase assays wereperformed as described in Example 9. The results are presented in Table4.

TABLE 4 Luciferase Presence of Activity Chimeric Receptor RH5992 (lightunits) 35S/GAL4-EcR³³⁰⁻⁸⁷⁸ −  2,804 35S/USP-VP16 −  6,12135S/GAL4-EcR³³⁰⁻⁸⁷⁸ + 35S/USP-VP16 −  3,586 35S/GAL4-EcR³³⁰⁻⁸⁷⁸ +35S/USP-VP16 + 130,601

The above results show that the 5′ regulatory region of the targetexpression cassette comprising the GAL4 response elements can beactivated in plant cells by the receptor polypeptides GAL4-EcR³³⁰⁻⁸⁷⁸and USP-VP16 in the presence of a complementary chemical ligand. Thelevel of expression of the target polypeptide luciferase was 36-foldabove that observed in the absence of chemical ligand. This indicates 1)that the chimeric receptor polypeptide bound to the GAL4 responseelements of the target expression cassette, 2) that RH5992 bound to theligand binding domain of the EcR³³⁰⁻⁸⁷⁸ moiety in the chimeric receptorpolypeptide, 3) that the two chimeric receptor polypeptides properlyheterodimerized, and 4) that the heterodimerization brought thetransactivation domain from VP16 into position for activation.

Example 14 A Transactivation Domain Can be Used on Each ChimericReceptor Polypeptide

Using the transformation method of Example 9, the receptor expressioncassette 35S/GAL4-EcR³³⁰⁻⁸²⁵-C1 (Example 6), the receptor expressioncassettes 35S/USP-VP16 or 35S/VP16-USP (Example 4) and the targetexpression cassette (GAL4)₁₀-Bz1/Luc (Example 7) were co-transformedinto maize cells. Transformed cells were incubated in the presence of 10μM RH5992 as chemical ligand for approximately 48 hours. Luciferaseassays were perfomed as described in Example 9. The results arepresented in Table 5.

TABLE 5 Luciferase Presence of Activity Chimeric Receptor RH5992 (lightunits) 35S/GAL4-EcR³³⁰⁻⁸²⁵-C1 + 35S/VP16- −  3,423 USP35S/GAL4-EcR³³⁰⁻⁸²⁵-C1 + 35S/VP16- + 11,069 USP 35S/GAL4-EcR³³⁰⁻⁸²⁵-C1 +35S/USP-VP16 −  5,972 35S/GAL4-EcR³³⁰⁻⁸²⁵-C1 + 35S/USP-VP16 + 37,579

The above results show that the 5′ regulatory region of the targetexpression cassette (GAL4)₁₀-Bz1/Luc comprising the GAL4 responseelements can be activated in plant cells by the receptor polypeptides35S/GAL4-EcR³³⁰⁻⁸²⁵-C1 and 35S/VP16-USP or 35S/USP-VP16 in the presenceof a complementary chemical ligand. The expression of the targetpolypeptide was greater (6-fold over the absence of ligand) with VP-16fused to the C-terninal end of USP compared to fusion of VP-16 to theN-terrminal end (only a 3-fold enhancement).

Example 15 Isolation of Receptor Polypeptide Mutants having LoweredBasal Activity

Mutations in the ligand binding domain of both the ecdysone receptor(EcR) or the Ultraspiracle receptor (USP) were generated in vitro usingPCR mutagenesis as described by Leung et al., Technique 1: 11-15 (1989).PCR fragments of mutated EcR ligand binding domain were cloned into ayeast expression vector as a fusion with the DNA binding domain of yeastGAL4. PCR fragments of mutated USP ligand binding domain were clonedinto a yeast expression vector as a fusion with the transcriptionalactivation domain of VP16. Mutant constructs were transformed into theyeast GAL4 reporter strain GGY1:171. The mutant GAL4-EcR constructs weretransformed in combination with non-mutagenized VP16-USP, and mutantUSP-VP16 constructs were transformed in combination with non-mutagenizedGAL4-EcR³³⁰⁻⁸²⁵-C1. Yeast transformants were plated on media containingthe indicator X-Gal. Mutants having a decreased basal level of receptorpolypeptide activity for the heterodimer generated white to light bluecolonies on X-Gal indicator plates, while the non-mutagenized receptorpolypeptide heterodimers generated dark blue colonies. White to lightblue colonies are tested for the basal and chemical ligand-induced levelof receptor polypeptide activity by growing yeast cells representingthose selected colonies in S media containing glycerol, ethanol andgalactose as carbon sources. The resulting culture is split into twoportions, one of which is treated with an appropriate chemical ligandand the other is used as a control in the absence of chemical ligand.After exposure to the chemical ligand, both the treated and controlportions of the culture are assayed for β-galactosidase activityaccording to the procedure of Miller (Experiments in Molecular Genetics,p. 352-355, J. H. Miller, Ed., Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1972). The nucleotide sequences which encode themutant receptor polypeptides isolated and identified by this techniqueare candidates for further testing since they may exhibit, in plantcells, a decreased basal activity and a greater fold induction of targetgene expression in the presence of the chemical ligand.

Example 16 Identification of Mutant Receptor Polypeptides with ImprovedFunction in Plant Cells

Receptor expression cassettes which encode the mutated EcR or USPreceptor polypeptides of Example 15 are prepared according to the aboveExamples 1 through 6. These receptor expression cassettes, incombination with one of the target expression cassettes of Examples 7and 8, are transformed into plant cells according to the procedure ofExample 9. Transformed plant cells are tested for activation of the5′-regulatory region of the target expression cassette by the mutantreceptor polypeptides in the presence of an appropriate chemical ligand.Mutant EcR or USP receptor polypeptides which produce, in plant cells,low basal expression of a target polypeptide in the absence of chemicalligand and high expression of target polypeptide in the presence of anappropriate chemical ligand are useful for controlling gene expressionin plants.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example four purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A receptor expression cassette for use in a method of controllinggene expression in a plant, comprising: (a) a 5′ regulatory regioncapable of promoting expression in a plant cell; (b) an operably linkedcoding sequence encoding an Ecdysone receptor polypeptide comprising aligand binding domain and a DNA binding domain, and a heterologoustransactivation domain, wherein said heterologous transactivation domainis the transactivation domain from the C1 regulatory gene of maize; and(c) a 3′ terminating sequence.
 2. A plant transformation vectorcomprising the receptor expression cassette of claim
 1. 3. A plant celltransformed with the plant transformation vector of claim 2.